This proposal examines the molecular basis of differential gene expression in yeast (Saccharomyces cerevisiae). Yeast has three cell types -- a, Alpha, and a/Alpha -- that produce many distinct proteins, but are determined by alleles of single genetic locus, MAT. The structural genes for these cell type-specific proteins appear to be unlinked to MAT but their expression is differentially regulated by alleles of the mating type (MAT) locus. MATAlpha, for example, is proposed to code for two functions: a positive regulator of unlinked Alpha-specific genes (Alpha1 and a negative regulatior of unlinked a specific genes (Alpha2). Our genetic analysis has identidied an unlinked Alpha-specific gene (STE3) and an unlinked a-specific gene (BAR1) that are likely regulated by MATAlpha. Our goal is to determine the molecular basis of regulation of these genes by MAT. To understand how cell type-specific gene expression occurs, we have isolated the STE3 gene by molecular cloning techniques, and have shown that an RNA transcript complementary to this clone is found in Alpha cells, but not in a or a/Alpha cells. We will use in vitro and in vivo mutagenesis to identify regions near the STE3 gene which are required for its Alpha-specific expression. We will also isolate mutations that alter the regulation of this gene, for example, mutations that cause its expression in other cell types. These mutations may be either in regions near STE3 or in other genes whose products are required for STE3 expression. We will also clone the a-specific BAR1 gene and analyze how its expression is negatively controlled by Alpha2.